Diabetic nephropathy is characterized by glomerular abnormalities leading to end-stage renal failure in 35% of patients with type I diabetes. Extensive fibrosis of the renal interstitium, however, as is seen in many renal diseases, is the best structural correlate of the failing kidney in diabetes. Nephromegaly is also a characteristic of the kidney in diabetes and may herald later development of renal insufficiency. In this proposal, we will seek to examine the mechanisms whereby hyperglycemia produces increased extracellular matrix and ultimately fibrogenesis as well as increased kidney size, a factor which may contribute to abnormal extracellular matrix formation. Preliminary data which we have gathered suggest that a transformed cell line derived from mouse cortical tubular epithelium represents an excellent model system for evaluating the effects of high glucose on extracellular matrix formation. In this system, raising medium glucose from 100 to 450 mg% stimulates both type I and type IV collagen secretion as well as increases steady state messenger RNA levels for these collagen molecules. In addition, high extracellular glucose suppresses cell proliferation yet induces cellular hypertrophy in vitro. This proposal seeks to expand on these initial observations to develop information on the mechanisms whereby high glucose levels ultimately result in renal fibrosis and renal failure in diabetes. We will examine the direct effects of high glucose both in vivo by utilizing histomorphometry, fluorescence microscopy, and in situ nucleic acid hybridization, as well as in vitro by using radioimmunoassays for collagen secretion and a variety of assays for collagen gene expression in order to understand the mechanisms of the glucose-induced effect. In addition, we will examine the effects of high glucose levels on the response to various growth factors in cell culture. In particular we will explore the autocrine function of Transforming Growth Factor-beta as a potential mediator of some of the effects of elevated glucose concentrations. We have also found in preliminary data that polyol pathway activation, an important cause of the early functional abnormalities in diabetic nerve and retina and possibly kidney is important in our in studies of increased extracellular matrix formation since aldose reductase inhibition or myo-inositol supplementation reverses high glucose-induced excess types I and IV collagen synthesis. Our in vitro studies will be performed on two cell types residing in the renal cortical tubulointerstitium, proximal tubule cells and renal fibroblasts. The results of these studies should provide new insights into the effects of hyperglycemia on the ultimate progression of renal failure in diabetes with a particular emphasis on the effects of altered matrix synthesis.